Bull. Org. mond. Santw Bull. Wid. Hlth Org. 1964, 31, 539-544

Laboratory Culture of , , and Stomoxys

H. F. SCHOOF 1

In spite of great improvements since 1940, diffi- Larvae culties are still encountered in rearing the large Various media have been used for rearing: horse numbers of of uniform quality required by manure (Glaser, 1924), horse manure and additives modern entomological research. These difficulties (Glaser, 1927; Hockenyos, 1931), and cow manure partly account for variations in techniques, rearing (Feldman-Muhsam, 1944). Richardson (1932) pro- media and equipment. posed the alfalfa-meal/wheat-bran/yeast formula that was the forerunner of the current CSMA mash REARING MUSCA (i.e., alfalfa meal 27%, wheat bran 33%, Brewer's dried grain 40%) and of the CSMA medium (Soap Musca domestica, the , because of its Blue Book, 1960), which consists of 340 g of the ease of handling and ability to propagate in widely CSMA mash mixed with 750 ml of aqueous sus- varying conditions, is probably the most pension that contains 15 g of moist yeast cake and commonly used for evaluation of insecticides. This 10 ml of diamalt. As usually occurs, this standard fact, together with the tendency to use different preparation 2 has been modified by various labo- methods in the various laboratories, either through ratories according to their own needs or desires choice or because of factors such as space, money, (WHO Expert Committee on Insecticides, 1961; and volume of required, has led to the use of Gahan, 1963; Basden, 1947). The YMA medium a number of slightly different techniques. (WHO Expert Committee on Insecticides, 1961; Rockstein, 1957) consists of dried yeast, 100 g; Eggs full-cream dried milk, 100 g; agar, 20 g; and boiling Housefly eggs are deposited in clusters or masses water, 1 litre. Larvae have been reared on cotton- upon substrates placed in the colony cages for that wool pads soaked with diluted milk (milk: water, purpose, e.g., a cellulose cotton pad 3 inches (7.5 cm) 3: 1) (Hafez, 1948) and on fermenting dog-biscuits in diameter and 1 inch (2.5 cm) thick and soaked in (Frings, 1948). milk and honey-water,2 dental cotton-wool rolls For , Fales (1963) found cow impregnated with a weak solution of milk powder manure to be the only medium that sustained the and dried yeast (Sawicki and Holbrook, 1961), larvae to maturity. In India, coconut poonac is an crimpled whole oats (Eagleson, 1937), filter-paper excellent substrate for M. d. vicina (Tharumarajah & soaked with milk and a few drops of ammonia Thevasagayam, 1961); in Japan a mixture of (Okara) (Fisher & Morrison, 1949), fermenting dog-biscuits tofu, 50 g, rice bran, 5 g, and yeast powder, 0.5 g (Frings, 1948), and numerous other materials (Soap is used (Nagasawa & Hashizume, 1955). Other Blue Book, 1960). After 18-24 hours the eggs hatch media for M. domestica have included crimpled when the pads are moistened with or immersed in whole oats, syrup and yeast (Eagleson, 1937), water.2 Egg quantity can be calculated volumetri- guinea-pig meal (Hampton, 1952), coffee-grounds cally, 500 (West, 1951) to 750 (Smith and Harrison, and cheese (Hase, 1935) and tissue-paper, milk 1951) per 0.1 ml or weighed (1100 newly-hatched powder and yeast (Spiller, 1963). Media are hand- larvae = 90 mg). mixed or mechanically mixed; the main point is to mix thoroughly to ensure uniform moisture distri- bution. The finished medium 2 is 24 hours 1 Assistant Chief, Biology/Chemistry Section, Technology prepared Branch, Communicable Disease Center, Public Health in advance of use and 2 pounds (900 g) are placed Service, US Department of Health, Education, and Welfare, in a plastic bag that is inserted in a 1-US-gallon Savannah, Ga., USA. ' Unpublished memoranda of the Communicable Disease (3.7-litre) battery jar. Packing the medium is Center Laboratories, Savannah, Ga. avoided. Newly-hatched larvae are placed on the 1507 -539- 540 H. F. SCHOOF medium and 3-4 days later a 3-inch (7.5-cm) layer supply of water (Gahan, 1963); or agar, banana, of sand is added.' Other seeding rates are 5000 eggs sugar, skimmed milk, formol, gelatin and water in 10 litres of medium (Sawicki & Holbrook, 1961) (Eagleson, 1943). Milk in liquid or dry form is and 2000 eggs per 75 g of a dry mix of milk powder, essential to flies used for egg production but not to absorbent tissue-paper and yeast with 2.1 ml water those used for test purposes. per g of mix (Spiller, 1963). For CSMA media, temperatures up to 130°F (54°C) are considered Adult colonies allowable. Odour from fermentation can be mini- In susceptible colonies 3000 pupae are placed in a mized by continually ventilating the rearing room. cage 14.5 x 16.5 x 19 inches (about 36 x 41 x 46.5 cm) and provided with sugar cubes, powdered milk, and Pupae water. An oviposition pad is put in the cage over- At 80°F (260C) and 70 % relative humidity, larvae night. Cages which provide less than 1 cubic inch begin to pupate in the upper portion of the medium of space per reduce the fertility and longevity -i.e., sand, medium (Soap Blue Book, 1960), of adults. At 80°F (26°C) Rockstein & Lieberman vermiculite, sawdust (Sawicki & Holbrook, 1961)- (1959) reported males to have a shorter life-span 4-5 days after the eggs are set. Pupation is usually than females (17 days, 3, 30 days ?). Normally, complete by day 7. The sand is then poured from begin oviposition on the third or fourth the jar through a sieve to remove the pupae.1 In the days after emergence, with the majority of eggs CSMA technique, the upper portion of the medium deposited within the first 10 days of oviposition is removed, poured into a tray and allowed to dry (Wilkes et al., 1948; Buei, 1959). Adults of M. in the air. autumnalis are confined in cage 1Ox 1Ox 10 inches Pupae are separated by directing an air current (25 x 25 x 25 cm) and fed skimmed milk: sugar against the medium-pupae mixture. The upper, (2: 1 v/v) and a small amount of diamalt solution puparia-laden, stratum also may be put in water, containing brain-heart infusion and pollen (Fales, where the medium sinks and the pupae float (Gahan, 1963). Citrated beef blood and cow manure are 1963). Born (1954) found sand to be a deterrent to also provided. Female M. autumnalis begin egg mould growth when a 1-inch (2.5-cm) layer was deposition at 6 days of age. Fresh cow manure added to the medium 48 hours after it had been serves as the egg-laying medium. M. autumnalis seeded. Puparia can be calculated in number either does not oviposit readily except in sunlight or high by weight or by volume. Normally, pupae have an artificial illumination. average weight of 18-22 mg and average 25 per ml.

Life-cycle FACTORS INFLUENCING FLY DEVELOPMENT At 80°F (26°C), the time sequences are: eggs Temperature, humidity and light hatch, 16-24 hours; larvae mature, days 3-4; pupae present, days 4-5; adults emerge, days 8-10; ovi- Generally, the period of development for each position begins, day 12 or 13. stage decreases as the temperature rises. At 74°F (24°C) the eggs of M. domestica required 17 hours Adult food and care before hatching whereas at 99°F (37°C) the period Each holding cage is supplied daily with one of was 7.6 hours (Davidson, 1944). Eggs of M. d. the following: a solution of 5 % spray-dried non-fat vicina hatched in 6 hours at 95°-100°F (35°C-38°C) milk with solids and 2% sugar (Soap Blue Book, and in 11 hours at 77°-86°F (25°C-30°C) (Hafez, 1960); dry powdered milk (Sawicki & Holbrook, 1941). Adult M. domestica are active from 44°F 1961); granulated sugar, liquid milk and 10% to 1 10°F (7°C-43°C) with the optimum at 92°F- sucrose; sugar cubes and water; 1 solid sugar and a 93°F (33°C-33.50C) (Nieschulz, 1935). At 60°F mixture of equal parts of cow's milk and water (15.5°C) adults survived up to 91 days (Dove, 1916). plus 1: 2000 parts of formol as a preservative; a dry The effect of humidity is usually combined with mixture containing 6 parts granulated sugar, 6 parts temperature. However, excessive moisture is detri- powdered milk, 1 part powdered egg and a separate mental to larval development. The significance of light in rearing procedures for M. domestica appears but with M. 1 Unpublished memoranda of the Communicable Disease minor, autumnalis strong artificial light Center Laboratories, Savannah, Ga. or sunlight is necessary for oviposition (Fales, 1963). LABORATORY CULTURE OF MUSCA, FANNIA AND STOMOXYS 541

Crowding length of adult stage, egg production or egg viability Too many larvae for a given supply of food results (Pimentel et al., 1951). No differences were noted in increased larval mortality and smaller adults. in six susceptible and two DDT-resistant strains in Adult crowding causes excessive activity, com- regard to egg viability, length of larval cycle or adult petition, decreased longevity, and greater mortality. production rate (Babers et al., 1953) but in Denmark An increase in the diameter of the rearing container the larvae of a DDT-resistant strain were found to from 4 cm to 7 to 10 cm resulted in the period develop faster than those of a susceptible one between oviposition and the emergence of female (B0ggild & Keiding, 1958). Changes in biotic M. d. vicina declining from 17.3 to 13.1 to 12.0 days potential and/or resistance levels may also be the (Nagasawa & Kishino, 1959). result of differences in handling procedures (McKenzie & Hoskins, 1954). Quality offood The addition of small amounts of vitamin A, REARING FANNIA thiamin, riboflavin, panthenic acid, pyridoxine, and Fannia canicularis has not been reared extensively RNA to fly media was not beneficial, but at high but interest in its culture has arisen recently because dosages they were harmful (Sawicki & Holbrook, of the nuisance created by it in homes and dairies 1961). Cholesterol also had no effect. M. domestica in various parts of the United States of America. has been reared on a synthetic medium (Monroe, 1962) and the weights of larvae, pupae, and adults Eggs approached those of flies reared by the CSMA of Eggs of F. canicularis are deposited on chicken method. Acidity (Meister, 1962), amounts yeast, or pigeon faeces, or on cellulose cotton pads soaked and ratio of water to media (Hirakoso, 1962) with sour milk (Fay et al., 1963) or with 4.5% w/v influence production. protein hydrolysate (Lewallen, 1954). Aging of the Biological state protein hydrolysate pads for one or two days increases the suitability of this substrate. Steve (1960) The age of the female at the time a batch of eggs found that CSMA mash moistened with household is laid influences the percentage hatched and the ammonia water (1: 1) is suitable and that chicken longevity of adults in succeeding generations excreta is the medium ofchoice as against hog, horse, (Meister, 1962). Any selection of a developmental cow and sheep manure. Davis (1963) used a 3-day- stage on a continuous basis will eventually segregate old alfalfa, meal, and water mixture or extract certain characteristics that could enhance or handi- therefrom. Egg collection is improved by placing cap the survival and propagation of the species. in a 0.5-US-pint (236-ml) carton a pad soaked with Another important biological characteristic is the protein hydrolysate and covered with protein- susceptibility level of the colony to insecticides. The hydrolysate-soaked paper towelling that is formed problem is to maintain uniform levels of suscepti- into a wrinkled lining for the carton (Fay et al., bility or resistance. Susceptible colonies are usually 1963). Eggs laid in the folds of the towelling are maintained by isolating the susceptible strains from flushed off by water and concentrated on a fine black the resistant colonies, by preventing insecticide con- batiste cloth in a funnel. With F. benjamini an ovi- tamination, or by both. With resistant colonies the position medium of a cellulose cotton pad soaked level of resistance to a particular chemical is main- with 5 % protein hydrolysate in normal saline can be tained by exposing the adult colonies to residues of used (Winkler & Wagner, 1961). that insecticide. Either a proportion I of or the entire (Gahan, 1963) cage surface is treated. Larvae Resistant strains of M. domestica have shown For larval culture 20 % molasses solution con- differences in the rearing cycle as compared to taining one yeast cake per 1000 ml can be added to susceptible strains. In a susceptible strain the peak CSMA mash just under saturation (Steve, 1960) or of pupation occurred on day 5; in a highly resistant 11 ounces (312 g) of mash can be mixed with 5 ml strain pupation was at day 9. No differences were of diamalt, 45 ml of yeast suspension, 0.5 pound apparent in pupal weight, pre-ovipositional period, (227 g) yeast cake/750 ml water and 1 litre of water (Fay et al., 1963). Lewallen (1954) used 112 g 1 Unpublished memoranda of the Communicable Disease Center Laboratories, Savannah, Ga. laboratory "chow ", 225 g all-wheat bran, 30 g 8 542 H. F. SCHOOF dried Brewer's yeast and 12.5 ml diamalt in 598 ml Adult food and care water. The maintenance of a higher water content Adult F. canicularis feed readily from cellulose in the medium than is normally present in housefly cotton pads soaked with a mixture of powdered media appears essential for successful development whole milk, 4 ounces (113 g) and extracted honey, (Fay et al., 1963). 5 ml in 1250 ml of water (Fay et al., 1963) and lump In the rearing procedure 0.5-1.5 inches (1.3-3.8 sugar and water (Fay et al., 1963). Separate 1: 1 cm) of the substrate are placed in a battery jar, solutions of molasses-water and of evaporated-milk/ crystallizing dish or other suitable container, just water (Steve, 1960) or sugar water (20%) and under the surface of the medium. Fay et al. (1963) protein-hydrolysate water solution (4%) (Lewallen, seeded each container (150 mm diameter, 75 mm 1954) are also used. F. benjamini is apparently more depth) with approximately 1000 eggs (50 mg), while sensitive to deprivation of carbohydrate than of Lewallen (1954) used 400-600 eggs per US gallon water, as complete mortality occurred with flies (3.78 litre) container. Newly hatched larvae of given only water while those provided with sugar F. benjamini feed on the protein hydrolysate in the survived (Winkler & Wagner, 1961). oviposition pad for several days, after which canned milk is added to the pad (Winkler & Wagner, 1961). Adult colonies Pupae Colony cages, 14 x 18 x 12 inches (35 x 45 x 30 cm) At 75F-80°F (24°C-26.50C) larval development are stocked with 3000 pupae initially and supple- of F. canicularis is completed within 8-10 days, the mented by weekly additions of 1000 each. Ovi- specimens moving to the top of the medium for position occurs within 4-6 days after emergence and pupation. The placing of corrugated cardboard continues up through 38 days. Approximately 50% strips on the medium causes 95 %-98 % of the larvae of the eggs are produced by day 20 and 90% by to within the and day 30. A cyclic trend in oviposition, with peaks pupate strips greatly reduces the when the females are 7, 11, 16-17, and 24 days old, time spent in removing pupae from media (Fay et al., has been reported et 1963). The strips are easily removed and the pupae (Fay al., 1963). then released by ripping the corrugated portion from its backings. Pupae average 7 mg in weight, REARING STOMOXYS CALCITRANS and 1000 represent a volume of 27 ml. With F. ben- jamini the pupae-covered rearing pads are stored Stomoxys calcitrans, the stable-fly, is considered under a glass container and sprinkled daily with less amenable to laboratory rearing than is the distilled water (Winkler & Wagner, 1961). housefly. Life-cycle Eggs At 75°F-80°F (24°C-26.5°C) the sequence of S. calcitrans lays eggs readily on damp cellulose development of F. canicularis is: cotton pads placed in a dish containing citrated bovine blood, 64 ml of 5 % sodium citrate in 1 litre Fay et al. Steve Lewallen (1963) (1960) (1954) of blood (Campau et al., 1951), on water-soaked Eggs hatch (hours) 24-36 36-48 36-48 sponge or cotton (Champlain et al., 1954), or in the Larvae mature (days) 7-9 8-10 8-10 CSMA medium (Doty, 1937). In East Africa, Parr Pupae present (days) 8-10 9-10 8-12 (1959) used a fermented mixture of dried cow dung, Adults emerge (days) 19-29 18.5-22 19-24 9 ounces (255 g), dried bullock blood granules, Oviposition begins (days) 25 23.5-27 24-29 5 ounces (142 g), and sugar, 1 ounce (28 g). Ovi- With F. benjamini the eggs hatch in 1-3 days, position will occur on cotton moistened with 0.1 % larval development requires 5-7 days, and the egg-to- ammonium hydroxide (Woodbury, 1943) or on adult period is slightly longer than 15 days (Winkler black cloth wrapped around moist cotton slightly & Wagner, 1961). Production methods for F. cani- dampened with 5 % ammonium hydroxide cularis are relatively inefficient as only one-third of (McGregor & Dreiss, 1955). The moistened sponge the larvae form pupae and of these 70% develop method (Champlain et al., 1954) is an advantage to the adult stage (Fay et al., 1963). Steve (1960) since the eggs can be flushed from the sponge by found that half of the females died within 24 days immersing it in water and squeezing in a tray. The but Fay et al. (1963) reported 50 % survival at dislodged eggs sink and the excess water can be 35 days. poured off. Where the number of eggs is not to be LABORATORY CULTURE OF MUSCA, FANNIA, AND STOMOXYS 543 determined, the oviposition media are frequently present, day 9-12; adults emerge, day 14.5-16; placed directly into the larval rearing medium oviposition begins, day 21.5. Campau et al. (1951) (Doty, 1937; Parr, 1959). reported adult yields of 57% from the quantity of eggs set. Emergence from pupae averaged Larvae 86%. Doty (1937) added oat hulls, 1 pound (0.45 kg) to wheat bran, 8.75 pounds (3.9 kg); alfalfa, 6.5 Adult food and care pounds (2.9 kg); water, 14 US quarts (13.2 litres); Citrated bovine blood provides a suitable source diamalt, 2 US fluid ounces (59.14 ml) and 1.5 US of nourishment for the adult flies. Warm blood pints (0.71 litres) of yeast suspension to produce a (Doty, 1937) is unnecessary since adults feed readily suitable medium. The standard CSMA medium was on ice-cold blood (Campau et al., 1951; Woodbury, used (Campau et al., 1951) while Parr (1959) em- 1943). The blood may be poured over cellulose ployed a cow dung/dried blood/sugar mixture. cotton pads in a dish (Campau et al., 1951), con- McGregor & Driess (1955) added 5 parts of wood tained in glass tubes (6-8 cm length) with open ends shavings to one part of CSMA mash but omitted (Eagleson, 1943) or used to soak absorbent pads yeast and diamalt. Goodhue & Cantrel (1958) mixed hung from the top of the cage (Parr, 1959). It may the mash (182.8 g) plus 20 % yeast suspension be necessary to replenish the blood supply twice (232 ml), diamalt (32 ml), water (3892 ml) with daily. Starved adult S. calcitrans (8.6 mg) are re- vermiculite (400 g in 2000 ml of water). Gingrich ported to consume three times their body-weight (1960) developed a wholly synthetic medium for of blood (25.8 mg) (Parr, 1962). Bovine blood is the aseptic larval rearing that produced a 72% yield common type used but that from horse or pig is of adults as compared to 88 % for the CSMA equally suitable (Pospisil, 1961). When fed on sheep, procedure. On the sterile diet adults emerged in guinea-pig, or mouse blood, females do not lay 27.6 days, on the CSMA medium emergence was in eggs. Others workers have indicated similar failure 14.2 days. with pig blood. Eggs are seeded at the rate of 0.5 ml per jar (Champlain et al., 1954) or 0.3 ml per jar (Campau Adult colonies et al., 1951), or the egg pad is placed directly on the larval medium. Cages, 30 x 30 x 28 inches (75 x 75 x 70 cm), hold 4000 to 8000 adults (Campau et al., 1951) but Doty Pupae (1937) kept only 50 to 200 specimens in a cage After 9-11 days the majority of the larvae form 18x12xll inches (45x30x27.5 cm). In cages puparia. Removal of the hard crust on the top of similar in size to Doty's, Campau et al. (1951) the CSMA mash shows the pupae concentrated on reported lowered egg production. However, Cham- the surface of the medium near the periphery plain et al. (1954) reported egg production in a cage (Campau et al., 1951). Another technique is to put 14x12x12 inches (35x30x30 cm), equal to that a half-inch (1.3 cm) layer of sand on the surface of in a cage 30 x 30 x 28 inches (75 x 75 x 70 cm). the medium, keep it moist from the 7th day after Cylindrical cages 14 inches diameter, 22 inches long seeding until the first adults emerge, and then place (35 cm diameter, 55 cm long) were used to hold the puparia-laden sand in water to allow the puparia 2000 flies (Jones, 1963). to float free (Champlain et al., 1954). With the Adults mate at 6 days of age and oviposition cow-dung/blood/sugar medium (Parr, 1959), the begins one day later (Eagleson, 1943). Where the larvae move to the top layer (surface to 1 inch, citrated blood is poured over a cellulose cotton pad 2.5 cm, deep) of the medium and this layer is re- in a dish, egg laying usually begins on the cellulose moved and the puparia collected by use of forceps. cotton when the level of blood in the dish drops Puparia average 11-12.6 mg in weight (Campau (Campau et al., 1951). The female produces 10 or et al., 1951; Parr, 1962). 11 batches of eggs, each averaging 35.5 eggs. A single mating appears sufficient. The number of Life-cycle eggs per batch shows no decline with age of the At 80°F (26.5°C) the time sequences are: eggs female and the level of viability remains similar hatch, 24-36 hours; larvae mature, day 8-11; pupae (Eagleson, 1943). 544 H. F. SCHOOF

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